Biochemistry February 25, 1975
Volume 14, Number 4
@ Copyright 1975 by the American Chemical Society
Environment and Conformation Dependent Sensitivity of the Arsanilazotyrosine-248 Carboxypeptidase A Chromophoref Jack T. Johansen and Bert L. Vallee*
ABSTRACT: Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex with the active site zinc atom. Arsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum a t 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum a t this wavelength, also characteristic of this complex. Both spectra a r e exquisitely responsive to p H changes and serve to monitor formation and dissociation of the metal-azopheno1 complex. Two pK,,, a t 7.7 and 9.5 delineate the p H range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazocarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr248 residue away from the zinc atom. The now available chemical, functional, and structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanila-
zo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its G l y - ~ - T y rcomplex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248 Zn interaction now leads to the conclusion that in up to 25% of the molecules in the crystals Tyr-248 interacts with the active site zinc atom (W. D. Lipscomb (1973), Proc. Nut. Acad. Sci. 1i.S. 70, 3797). Thus, even in the crystals the enzyme exists in a t least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of G l y - ~ - T y rand P-phenylpropionate on solutions of arsanilazo- and of 'nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of Tyr-248 to the zinc atom, when altered by substrates and inhibitors, may reflect certain of the properties characteristic of the entatic, active site.
c h e m i c a l modification of carboxypeptidase A with diazotized arsanilic acid has successfully served a number of experimental objectives. In crystalline carboxypeptidase this reagent couples selectively with Tyr-248, a residue in the active site of the enzyme. The resultant product retains both peptidase and esterase activity (Johansen and Vallee, 197 1, 1973; Auld and Holmquist, 1973). Further, absorption and circular dichroic spectra of such azochromophores have proven remarkably effective in probing the local and overall
conformation of carboxypeptidase A (Vallee et ul., 1971), procarboxypeptidase (Behnke and Vallee, 197 l ) , and carboxypeptidase s, the product of proteolytic cleavage with subtilisin (Riordan and Livingston, 197 1). The absorption maxima between 330 and 390 nm and that a t 485 nm are characteristic of the protonated and ionized azoTyr-248 species, respectively, and the corresponding circular dichroic bands reflect local asymmetry. But most importantly, the yellow arsanilazotyrosine-248 moiety of carboxypeptidase forms a characteristic red chromophore with an absorption maximum a t 510 nm by complexing with the zinc atom present a t the active site of the enzyme. In solutions of zinc arsanilazotyrosine-248 Carboxypeptidase' the azophen-
~~~
From the Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 021 15, and the Carlsberg Laboratory, Chemical Department, Copenhagen, Denmark. Received September 6 , 1974. This work was supported by Grants-in Aid G M - I 5 0 0 3 and GM-02123 from the National Institutes of Health, of the Department of Health, Education and Welfare.
-
'
In order to simplify presentation, zinc carboxypeptidase, zinc arsanilazocarboxypeptidase, the zinc azoenzyme and apocarboxypeptidase, BIOCHEMISTRY, VOL.
14,
hO.
4, 1 9 7 5
649
JOHAhUSEN A N D
01 ligand and zinc are suitably juxtaposed to allow formation of an intramolecular azophenol Zn coordination complex which is optically active due to vicinal features and/or inherent asymmetry of the complex. However, removal of zinc or changing the mutual orientation of or distance between the zinc atom and the azoTyr-248 residue, e . g . , by crystallizing the enzyme, abolishes the spectral manifestations of the complex, which probes the vicinity of its constituents in an "all or none" manner (Johansen and Vallee, 197 1, 1973). This intramolecular coordination complex also responds to environmental factors, such as hydrogen ion concentration, substrates and inhibitors, denaturing agents, and the physical state of the enzyme which affect its conformation-dependent functional properties. The chromophore, moreover, is well suited to studies of steady state and transient state catalysis and to the examination of the effects of spectral perturbants such as substrates, pseudosubstrates, and inhibitors both by kinetic and equilibrium methods (Johansen and Vallee, 197 I ) . The properties of this intramolecular azoTyr-248 Zn carboxypeptidase coordination complex are closely similar to those of azophenol metal complex ions which serve as models and define the chemical basis of the spectral changes. Azophenols have not been used widely as reagents for the study of structure-function relationships in enzymes, and the chemical basis of their attractive spectral properties has not been discussed frequently in the biochemical literature. However, 2 decades ago Klotz emploled ternary azopyridine-metal-protein complexes as model systems for metal-catalyzed enzymatic reactions (Klotz and Ming, 1953; Hughes and Klotz, 1956), and his earlier conclusions regarding the properties of azophenol metal complex ions are quite analogous to those reached based on the observations to be presented. We here report the pH dependence of spectra of zinc azocarboxypeptidase, apoazocarboxypeptidase, and of two azophenols, arsanilazo-~l'-acetyltyrosineaniideand tetrazolylazo-.l'-carbobenzoxytyrosine (TAT) and its complexes with Z n J k , C'd'+, Hg'+. and Mn2+. I n addition, circular dichroic and absorption titrations of the zinc azoenzyme with glycyl-1,-tyrosine and P-phenylpropionate demonstrate the probe characteristics of the azochromophore. The data suggest that the spectra of azophenol metal complex ions and inti amolecular complexes can be effective prohes to examine local structure--function relationships of metalloenzymes.
-
-
Materials and Methods Carboxypeptidase A,, isolated by the method of Anson (19371, was obtained as an aqueous crystal suspension (Worthington Biochemical Corporation). Carboxypeptidase A,, was isslated by DEAE-cellulose chromatography from acetone powder of bovine pancreas according to the method apua7oc;irbox) peptidase. a n d apoiirsdnilazocarboxypeptidase are used interchangeabl) with zinc inonoarsanilazor~rosine-248 carboxgpeptidase and apodrsanilazot) rosine-218 carboxypeptidase, respectively. of ,in! enzynie form. Carbox>peptidasr A is referred to as native carboxypeptidase The enzyme einploqed for X-ray btructure anallsis with ii crJstal habit elongated 'ilong the a axis is designated as the X-ra) cr)st a l s A ~ o T > r - 2 1 8refers to t h e azophenol of monoarsanilazotSroh i n c . 2 4 8 and the azophenolate tu its ionized species. The absorption a n d circular dichroic spectra with an abhorption maximum and a negat i v c circulzr e x t ~ m u i nJ t 510 nm, respectivel~.are defined 3s the "red" ~ntramolecularcomplex. .At pH 8.5 and in the absence of t h e absorption band at 510 n m the bpectrum is defined as "yelloa." TAT. tetraioi)la7u-~~'-cd1.bob~nrox!t~rusine;DHT. diazonium-IH-tetra/ult'. T\ Lf. tet~aiiitroiiiethane.
VALLEE
of Cox rr ul. ( 1964) or, alternately, was obtained as a crystal suspension (Sigma Chemical Company). In all instances the crystals were washed three times with metal-free distilled water and recrystallized before use. Zinc arsanilazocarboxypeptidases were prepared and characterized as described (Johansen and Vallee, 1971, 1973; Johansen et al., 1972). Apoarsanilazocarboxypeptidase was prepared by soaking crystals in I , IO-phenanthroline (Auld and Holmquist, 1974). Protein concentration was measured by the absorbance a t 278 nm, based on a molar absorptivity a t 278 nm of 6.42 X lo4 M-' cm-' (Simpson et a[., 1963) for native carboxypeptidase, and 7.32 X IO4 M - ' cm-' for zinc arsanilazocarboxypeptidase (Johansen and Vallee, 197 1 ). Monotetrazolylazo-N-carbobenzoxytyrosine was prepared and purified as described by Sokolovsky and Vallee (1966). Its concentration was determined from the absorbance a t 416 nm, using a molar absorptivity of 4.39 X I O 3 M-' cm- I . /3-Phenylpropionate was recrystallized from water. Glycyl-L-tyrosine and guanidinium hydrochloride were purchased from Mann Research Laboratories, Inc., Z n S 0 4 * 5 l - I 2 0 , C d S 0 4 8 H 2 0 , M n S 0 4 5 H 2 0 , and HgC12 were "specpure" reagents from Johnson Matthey Co., Ltd. Stock solutions of each salt in metal-free distilled water were made up to a concentration of l o p 3 M. All other chemicals were reagent grade. All buffers used were freed of trace metal contamination by extraction with 0.1% dithizone in carbon tetrachloride (Thiers, 1957). Glassware and cuvets were cleaned by soaking in 1 : 1 nitric and sulfuric acids, followed by rinsing i n metal-free distilled water. Absorbance measurements a t single wavelengths were obtained with a Zeiss P M Q I 1 spectrophotometer. Absorption spectra were obtained with a Cary Model 14R or Model 1 18C spectrophotometer. For spectrophotometric pH titrations the thermostated Auld-French titration cell was utilized (Auld and French, 1970). The zinc- and apoarsanilazoenzynie, about 0.06 mM in 2 mM Tris-HCI-0.5 M NaCl buffer (pH 6.2) were titrated with aliquots of 0.1 r~ N a O H to result i n pH increments of 0.2-0.4. The absorption spectrum between 300 and 650 nm was recorded after each addition of base. Spectral titrations with glycyl-L-tyrosine and $-phenylpropionate were performed at pH 8.5 (0.05 M Tris-HCI-0.5 M haC1) by adding in a cuvet, microliter volumes of 0.1 M solution of the inhibitor or the substrate in the same buffer to 1.0-ml solution of the enzyme, 0.05 m ~ Results . were corrected for enzyme dilutions due to inhibitor or substrate addition. Spectrophotometric pH titration of T A T and of its metal ion complexes were performed as described for the enzyme. The T A T and metal ion concentrations were identical, i.e., 0.09 mM in the presence of 0.1 \I NaCI. Circular dichroism measurements were performed with a Cary Model 6 1 recording spectropolarimeter. All measurements between 300 and 600 nm were performed in I-ml quartz cells of 1.0-cm light path at enzyme concentrations ranging from 0.03 to 0.05 mM. Ellipticity is expressed as molecular ellipticity, [O]" = ( # / l O ) ( M / / c )with units of (deg cm')/dmol, where O = observed ellipticity in degrees; kf= molecular weight, (34,600 for carboxypeptidase A), I = path length in cm, and c = concentration in g/ml. I n accord with convention (Fairclough and Vallee, 1970), molecular ellipticities are not corrected for the refractive index of the solvent. The pH dependence of the circular dichroic spectra of zinc and apoazocarboxypeptidase was deter-
-
ENVIRONMENTAL SENSITIVITY OF
AZOTYR-248 CARBOXYPEPTIDASE
mined from p H 6.5 to 10.8 by stepwise addition of 0.1 N NaOH to 3-ml samples. The p H was measured before and after each spectrum was recorded. Circular dichroic titrations with glycyl-L-tyrosine and 0-phenylpropionate were performed as described for the absorbance titrations. Theoretical pH-titration curves were fitted to the experimental data by use of a nonlinear least-squares program written for the Hewlett Packard 98 10A calculator, and kindly provided by Dr. Thayer French. Sedimentation was carried out a t 59,780 rpm in a Spinco Model E ultracentrifuge. Sedimentation coefficients were corrected to viscosity and density of water a t 20'. Native and arsanilazocarboxypeptidase, 3.5 and 4.6 mg/ml, respectively, in 0.05 M Tris-1.0 M N a C l ( p H 7.5), and arsanilazocarboxypeptidase, 12.6 mg/ml in 0.1 M P-phenylpropionate-0.02 M Veronal-1.0 M NaCl (pH 7.5), were subjected to sedimentation. Results Reaction of carboxypeptidase A crystals with diazotized p-arsanilic acid uniquely modifies Tyr-248, as demonstrated by the presence of 95% of the label in C N B r fragment F I and by isolation, in 80% yield, of the duodecaptide corresponding to residues 246-257 of the primary sequence (Bradshaw et al., 1969; Johansen et a[., 1972). In solution, a t p H 8.2, this zinc azoenzyme is red. The peptidase activity a t p H 7.5, 0.05 M Tris-l .O M NaCl of this derivative is 60% and the esterase activity a t p H 7.5, 1 .O M NaCI, is 100% of that of the native enzyme, when Bz-Gly-Gly-L-Phe and Bz-Gly-Gly-L-0-Phe are the substrates, respectively. The absorption spectrum of the zinc azoenzyme has a maximum a t 510 nm (Figure 1A), characteristic of the formation of an intramolecular coordination complex between the azoTyr-248 moiety and zinc. Like other coordination complexes, the formation, stability, and dissociation of this azophenol metal complex and its spectra are sensitive to environmental factors, such as p H , substrates and inhibitors, the physical state of the enzyme, as well as any other conditions which can affect the ionization of the azophenol, modulate the conformation of the protein, or remove zinc. In the range of p H from 6.3 to 8.5 absorbance-pH titrations of zinc arsanilazoTyr-248 carboxypeptidase generate an absorption maximum a t 510 nm ( E 8000)2 with an isosbestic point a t 428 nm. On increasing p H to 10.8 the maximum shifts progressively to 485 nm ( t 10,500), characteristic of the free azophenolate ion, and two new isosbestic points become apparent a t 412 and 520 nm (Figure I A ) . The shift in A,, and the formation of a new set of isosbestic points demonstrate the existence of a t least three interconvertible species; that which predominates a t p H 6.1 is the protonated azoTyr-248, that a t -pH 8.5 is the intramolecular azoTyr-248 Zn complex, while that above p H 9.5 is the azoTyr-248 phenolate ion. Over the same p H range the spectra of the upoazoenzyme is quite different: the 510-nm absorption band is absent, but at p H 8.5 a maximum a t 485 nm becomes progressively prominent (Figure 1B). There is a single isosbestic point a t 416 nm, and the p H titration curve a t 485 nm fits a theoretical curve with a pK,,, of 9.4 (Figure l B , insert) and is consistent with two interconvertible species. T h e behavior is characteristic of the ionization of an azophenol. The titration is identical with that of monoarsanilazo-N-acetyltyros-
-
e
* The absorbance and
molar ellipticity values for the azoTyr-248.
Zn complex are measured at pH 8.5 and 23 f 0 . I o , those for the azophenolate ion at p H 10.8 and 23 f 0. I O
ineamide (vide infra) and of other, similar azotyrosine derivatives (Tabachnick and Sobotka, 1959). The insert of Figure IA shows the pH-titration curve of the zinc azoenzyme a t 560 nm, where the absorbance of the complex and that of the azophenolate ion differ maximally. In contrast to the typical sigmoid titration curve for the apoazoenzyme (Figure IB, insert) the titration curve for the zinc azoenzyme is bell-shaped with two pK values of 7.7 and 9.5, respectively; it is superimposable on a theoretical curve characterizing the formation and dissociation of a metal complex. Circular dichroic spectra can vary not only in amplitude but also in sign. In the present instance they resolve the spectral contributions of the complex from that of the azophenolate ion (Figure IC). A t p H 6.9 the circular dichroic spectrum exhibits two major Cotton effects, a negative band a t 340 nm ([elz3, -28,000), a positive band at 420 nm ([elz3, 14,000), and a negative band a t 510 nm. An increase in p H from 6 to 8.5 markedly intensifies the negative 510-nm extremum, concomitant with the decrease of the negative band a t 340 nm, while that a t 420 nm remains almost unaltered. These changes in circular dichroism correlate with the formation of the intramolecular azoTyr-248 Zn complex, as is evident from the absorption-pH titration (Figure 1A). A t p H 8.5 the value of the negative extremum a t 510 nm is maximal -43,000), but on increasing p H to 10.5 this is replaced by a positive extremum a t 485 nm ( [elz3,+10,500), characteristic of the azophenolate ion, while the 420-nm band shifts to 410 nm and becomes negative ( -4000). T h e circular dichroism-pH titration of the zinc azoenzyme a t 510 nm is bell-shaped and fits a theoretical curve with two pK,,, values of 7.7 and 9.5 (Figure 2), identical with those calculated from the absorbance-pH titration a t 560 nm (Figure IA). Though only 25 nm separate the maximum of the com510.nm) from that of the azophenolate ion (A,, plex (A, 485 nm), circular dichroism of the zinc azoenzyme can unambiguously distinguish between their extrema owing to their difference in sign. In accord with this, the apoenzyme completely lacks the negative extremum a t 5 I O nm over the entire p H range demonstrating that its presence is a direct function of the formation of the zinc complex (Figure ID). U p to p H 8 the apoazoenzyme exhibits only relatively small ellipticity bands a t 440 and 330 nm, respectively. As p H increases above 8.5, ionization of the azophenol generates a positive band a t 485 nm ([elz3, +10,500) and a negative band a t 410 nm ([elz3, -4000). A t p H I O the circular dichroic spectrum of the apoazoenzyme is identical with that of the zinc azoenzyme demonstrating that in both cases the uncomplexed azophenolate ion is the chromophore (Figures IC, D). Addition of Zn2+, Cd2+, Hg2+, or Ni2+ to the apoazoenzyme a t p H 7.5 forms the corresponding series of metallocarboxypeptidase with absorption maxima and circular dichroic extrema, characteristic for each metal. Titrations of each metalloenzyme reveal sets of two pK,,, values, typical for each particular metalloenzyme, as is apparent from the details of the absorption- and circular dichroism-pH titrations (Johansen et ai., 1973; Legg et ai.,to be published). O n standing for prolonged periods a t p H values above 10, the circular dichroic bands of the apoazoenzyme disappear, due to denaturation. The absorption-pH titrations of monoarsanilazo-N-acetyltyrosineamide as well as that of T A T and of its zinc complex closely resemble those of the apo- and zinc azoenzyme, respectively. Figure 3 shows the p H dependence of the visi-
+
BIOCHEMISTRY. VOL
14,
h O
4, 1975
651
JOHANSEN A N D VALLEE
+2
1.2
0
0.8
0.4
-2
W
n
u
C D U Y N
z
2
w
X
1.2
-4
v)
m
5, P
a
+1
0.8
0
0.4 -1
350
550
450
350
450
550
WAVELENGTH, nm F I G U R E I : Absorption and circular dichroic spectra of zinc and apoazoTyr-248-carboxypeptidase01. y. (A) Absorption spectra of the zinc azoenzyme at pH 6.2 (-), pH 8.3 (-), and pH 10.8 The insert depicts the absorption-pH titration curve a t 560 nm.(B) Absorption spectra of the apoazoenzyme at pH 7.5 (-) and at pH 9.9 (. * The insert is the pH titration curve at 485 nm. (C) Circular dichroism specpH 8.3 (-), and pH 10.8 (. . . .). (D) Circular dichroism spectra of the apoazoenzyme at pH 7.2 tra of the zinc azoenzyme a t pH 6.9 (-), (-) and pH 9.9 Enzymes were dissolved in 2 m v Tris-HCI-0.5 M NaCI. 23'. I n this and Figures 3 and 4 spectra for values of pH intermediate between pH 7 and pH 8.3 as well as between pH 8.5 and pH 10.5 have been omitted to simplify visualization of critical effects and changes. (e...).
a).
(e
e).
F I G U R E 2: Circular dichroism-pH titration curve of the zinc azoenzyme at 510 nm in 2 m\f Tris-HCI-0.5 I NaCI. 23'.
ble absorption spectrum of monoarsanilazo-N-acetyltyrosineamide. At low p H the spectrum of the protonated form exhibits an absorption maximum at 325 nm (e, 22,000), a shoulder a t 380 nm, and a broad envelope of overlapping bands extending to 560 nm. Increasing p H generates a new absorption band a t 485 nm (c, 10,5000) with an isosbestic point a t 41 5 nm. The absorbance-pH titration reflects a pK of 9.4 for the hydroxyl group of the azophenol (Figure 3, insert), and spectral properties characteristic both of monoazotyrosine derivatives and of apoazocarboxypeptidase (Figure I B and insert). Study of the spectral properties of bidentate azophenol-metal complexes, e.g., those of monoarsanilazo-N-acetyltyrosineamide Zn2+, presents prob-
-
652
BIOCHEMISTRY, VOL
14,
NO
4. 1 9 7 5
F I G L R E 3: Absorption spectra of monoarsanilazotyrosine amide at p t l 7.0 (-) and pH 13 The insert is the absorption pH titration curve at 480 n m . ( . . e
a).
lems due to their insolubility (Anderson and Nickless, 1967). However, tridentate heterocyclic and aromatic azophenols, exemplified by T A T , have proven particularly useful as metal indicators, owing to the solubility and high stability of their 1: 1 metal complexes (Anderson and Nickless, 1967; Johansen and Vallee, 1971). One of the azo nitrogens. the phenoxy group of the ionized azophenol, and the N-2 nitrogen of the tetrazolyl group are thought to serve as metal ligands.
ENVIRONMENTAL SENSITIVITY OF AZOTYR-248 CARBOXYPEPTIDASE
0.9
W
0.3
a
m
WAVELENGTH, nm 5: Effect of pH on the absorption spectrum of the tetrazolylazo-N-carbobenzoxytyrosine Cd complex ( 1 :1 ) in 0. I M NaCI. ( A ) Titration in the pH range from 5.0 to 8.3 where the complex forms, FIGURE
.
and (B) titration in the pH range from 9.2 to 11.2 where the complex dissociates. Numbers in the figure indicate the pH at which the spectra were recorded: (A) (1) pH 5.0, (2) pH 5.9, (3) pH 6.3, (4) pH 6.6, ( 5 ) pH 7.1, (6) pH 8.3. (B) (1) pH 9.2, (2) pH 10.2, (3) pH 11.2.
1
L
I I
I
I
I
I
I
I
450
550 650 WAVELENGTH, nm F I G U R E 4: Absorption spectra of ( A ) the tetrazolylazo-fi-carbobenzoxytyrosine Zn complex ( I : ] ) in 0.1 M NaCl at pH 4.3 (-), pH 8.5 (m), and pH 11.0 .). (B) Tetrazolylazo-N-carbobenzoxytyrosine in 0.1 M NaCl at pH 6.3 (-) and pH I I (. -). The insert iii (A) is the absorption-pH titration curve for the tetrazolylazoN-carbobenzoxytyrosine complex at 560 nm, and in (B) that for tetrazolylazo-N-carbobenzoxytyrosine alone at 482 nm. 350
-
( . . a
m a m
The p H dependence of the visible absorption spectrum of T A T (Figure 4B) is very similar to that of arsanilazo-Nacetyltyrosineamide (Figure 3); the pK of this phenoxy group is 9.0 (Figure 4B, insert). In the absence and presence of equimolar concentrations of Zn2+ ions a t p H 4.3 the spectra of T A T a r e identical (Figures 4A and B), since Zn2+ is not successful in dissociating the hydrogen ion of the azophenol a t this pH. As p H increases, Zn2+ ions displace this proton, generating an absorption band a t 510 nm which reflects the formation of the complex with isosbestic points a t 3 4 1 , 377, and 438 nm (Figure 4A). Further increase in p H to 11 progressively shifts the spectrum to that characteristic of the free azophenolate ion, ,,,A 482 nm, and new isosbestic points are formed a t 338, 398, and 497 nm. Substitution of Cd2+, Hg2+, or Mn2+ for Zn2+ generates complexes which exhibit absorption spectra typical for each metal ion and Figure 5 shows the detailed absorbancep H titration of T A T Cd2+ as a n example. While the results of pH-titration are similar for all the metals investigated, the absorption maxima, and the p H range where the complexes form and dissociate a r e characteristic for each particular metal (Table I ) . Figure 6 compares absorbancepH-titration curves for T A T in the presence of Zn2+,Cd2+, . ~ metal complexes dissoHg2+, and Mn2+, r e ~ p e c t i v e l y The ciate (or hydrolyze) in the p H range where the azophenolate ion forms. The change in absorbance as a function of
-
Oxidation of Co2+ to Co3+ precluded analogous titrations with this ion.
a
0.1
I
I
4
I
I
1
I
I
8
6
I
I
10
PH
6 : Absorption-pH titration curves of tetrazolylazo-"carbobenzoxytyrosine at 560 nm in the presence of Zn2+ (0),C d 2 + (+), Mn2+ (w), and Hg2+ (x), respectively, all in ratios of 1 : l in 0.1 M NaCI. FIGURE
p H is shown a t 560 nm, the wavelength a t which the absorbance of the Zn complex and that of the azophenolate ion differ maximally. The pH-titration curves of the metal Table I: Absorption Maxima and pKapp's Characterizing Formation and Dissociation of Metal Complexes of T e t razolylazo-N-carbobenzoxytyrosine. Maximum of
Complexing Metal Zn2+ Cd2+ Hg2+ Mn2+
Absorption (nm)
PK,,,"
PK,,,"
510
6.7
490
6.5
10.1 10.1
550
7.4
500
8.1
9.4 C
a pKappcharacterizing formation of the complex. pKapp characterizing dissociation of the complex. Not discernible under the conditions of the experiments.
BIOCHEMISTRY, VOL.
14, NO. 4, 1975
653
JOHAhSEN
AYD VALLEE
m
a m a 0
% +--
k
I j
7 -1
-4
i -1
i
1 11
1
350
I
,
I
450
I
I
I
I/
550
I
350
I
I
450
I
I I
I I
550
WAVELENGTH, nm
WAVELENGTH, nm
7: Effect of Gly-L-Tyr on the absorption spectrum (A) and circular dichroic spectrum ( B ) of zinc azoTyr-248 carboxypeptidase in 2 m v Tris-HCI-0.5 M NaCl (pH 8.5). Numbers indicate the G l y - ~ Tyr concentrations at which the spectra were recorded: (A) ( I ) 0; (2) 0.45 mu; ( 3 ) 1.7 n i M ; (4) 3.5 mM; (5) 11.1 m M .(B) ( I ) 0; (2) 0.4 mM; (3) 1.4 m M : (4) 3.8 mM: ( 5 ) 6.5 m M .
8: Effect of ,8-phenylpropionate on the absorption (A) and circular dichroic spectrum ( B ) of zinc azoTyr-248 carboxypeptidase i n 2 mM Tris-0.5 M NaCl (pH 8.5). Numbers indicate the B-phenylpropionate concentration at which the spectra were recorded: ( A ) ( 1 ) 0; (2) 1.2 mM; (3) 3.8 mM; (4) 6.2 mM; ( 5 ) 12 mM; (6) 50 mM. (B) ( I ) 0; (2) 1.5 mM; (3) 3.8 mM; (4) 6.5 mM; ( 5 ) 13 mM; (6) 50 m M .
complexes are bell-shaped. The ascending limb reflects the formation of the complex, as the metal ion successfully competes with a proton for the ligand. With increasing p H the competition of OH- with the azophenolate ion for the metal becomes apparent and absorption decreases. Thus, among other possible factors the steep slope of the descending limb of the M n 2 + titration curve a t high p H is due to precipitation of Mn2+ hydroxide. The effect of p H on the metal complexes in the present system are quite analogous to the behavior of other, similar azodye-metal complexes (Anderson and Nickless, 1967; Klotz and Loh Ming, 1953). The data closely resemble those of the apo- and zinc azocarboxypeptidase system and provide a chemical basis for their spectra. The series of chemical events which give rise to the spectral changes observed for the azoenzyme include the displacement of a proton from the azoTyr-248 to form the azoTyr-248 Zn complex and, a t higher pH, dissociation of the complex to result in free, ionized azoTyr-248, possibly due to competition with OH- ions (Figures 1, 4, and 6). Klotz and coworkers (Klotz and Loh Ming, 1954; Hughes and Klotz, 1956) came to similar conclusions based on their studies of a model system for metal-catalyzed enzymatic reactions, particularly of hydrolytic enzymes. Their studies of the effect of p I i on the formation of ternary proteinmetal-azopyridine complexe,s revealed that proteins such as serum albumin provide ligands to bind and thereby stabilize bidentate azopyridine-metal complexes. As a consequence, such mixed complexes form readily in the presence of proteins, just as observed in the present instance. Exposure of the zinc azoenzyme to 5 M guanidinium chloride abolishes the maximum a t 510 nm, and the absorp-
tion spectrum then becomes identical with that of the protonated azoTyr-248 moiety. Denaturation also completely abolishes all ellipticity bands of both the zinc and apoazoenzymes. Glycyl-L-tyrosine and a number of inhibitors also abolish the 510 nm band of zinc azoTyr-248 carboxypeptidase a (Johansen and Vallee, 1971). W e have now performed titrations with varying concentrations of Gly-L-Tyr and 8phenylpropionate a t p H 8.5, where the intramolecular coordination complex forms maximally. Gly-L-Tyr i s hydrolyzed a t about 5/1000th of the rate of Cbz-Gly-L-Phe and affects the absorption spectrum of the zinc azoenzyme in a manner almost identical with that of @-phenylpropionate (Figures 7A and 8A). Gly-L-Tyr in concentrations ranging from 0.2 to 10 mM abolishes the 510-nm absorption band of the intramolecular coordination complex while concomitantly increasing absorbance at 330 and 380 nm to that characteristic of the protonated azophenol (Figure 7A). Similarly, the amplitudes both of the negative and positive ellipticity bands a t 510 and 420 nm decrease, the latter shifting to 440 nm; there is a single, tight, isosbestic point at 445 nm (Figure 7B). The spectrum observed in the presence of 10 mM Gly-L-Tyr closely resembles that of the apoenzyme a t pH 8.5, unaltered even on increasing the concentration of the pseudosubstrate to 20 m M . The resultant absorption and circular dichroic titrations a t 510 nm result in superimposable, sigmoid titration curves with an apparent dissociation constant of 9 X lo-' M . Addition of /3-phenylpropionate in concentrations varying from 0.01 to 50 mM progressiveiy decreases the 510-nm absorption band of the complex while increasing those at 330
FIGURE
-
654
BIOCHEMISTRY. VOL.
1 4 , NO. 4, 1975
FIGURE
ENVIRONMENTAL SENSITIVITY OF
AZOTYR-248
and 380 nm with a tight isosbestic point at 428 nm. At a pphenylpropionate concentration of 50 mM, the spectrum becomes identical with that of the protonated azophenol (Figure 8A). In fact, overall, the changes in absorption as a function of both P-phenylpropionate and Gly-L-Tyr concentrations are virtually superimposable (Figures 7A and 8A) on those of the zinc azoenzyme as a function of p H (Figure 1 A). Concentrations of P-phenylpropionate up to 4 mM decrease both the amplitudes of the negative ellipticity band at 510 nm and that of the positive band a t 420 nm, with an isosbestic point at 450 nm. At yet higher concentrations the amplitude of both bands decreases further, but the isosbestic point gradually shifts to higher wavelengths. Furthermore, the extremum a t 420 nm gradually shifts to 380 nm, and a new negative band a t 450 nm is formed. These complex titration spectra suggest the presence of at least two modes of binding of P-phenylpropionate. The amplitudes of the circular dichroic and absorbance changes a t 510 nm on titration with P-phenylpropionate follow simple sigmoid titration curves with apparent dissociation constants of 3.1 and 3.2 mM, respectively. High concentrations of P-phenylpropionate are known to induce polymerization of native carboxypeptidase (Bethune, 1965a,b). Hence, their effect on the sedimentation of azoTyr-248 carboxypeptidase was examined. At 10 mM pphenylpropionate, where native carboxypeptidase exists as a polymer, the azoenzyme sediments as a single boundary with an ~ 2 0 =, ~3.3 S, a value identical with that for the native carboxypeptidase monomer. No evidence of dimers or higher polymers could be detected, analogous to results of comparable experiments with acetylcarboxypeptidase (Bethune, 1965b). Discussion Knowledge of the disposition of Tyr-248 with respect to the active site zinc atom of carboxypeptidase A is central to an understanding of the structure and function of the enzyme. A number of functional, chemical, and structural approaches have served to examine this problem. We here report the p H dependence of the spectra of the zinc and apoazoenzyme in solution, of the model, tetrazolylazo-N-carbobenzoxytyrosine and its complexes with Zn2+ and other metal ions and the spectral perturbation of the zinc azoenzyme by glycyl-L-tyrosine and P-phenylpropionate. We further relate and compare these results to all previous studies of this particular tyrosine and its spatial relationships to the zinc atom of the enzyme. Spectral Properties of Azochromophores. Selective coupling of diazonium salts with tyrosyl side chains of proteins generates intensely colored derivatives (Tabachnik and Sobotka, 1959, 1960) which exhibit distinctive circular dichroic spectra (Fairclough and Vallee, 1970). The location of the extrema, the signs, and the magnitudes of the resultant extrinsic Cotton effects are characteristic of both the diazonium salt and the protein and depend critically upon the relative concentrations of reactants, ambient conditions (Fairclough and Vallee, 1970), and the physical state of the protein (Vallee et al., 1971). These extrinsic Cotton effects reflect the stereochemistry of the azotyrosyl groups which are vicinally conditioned and, hence, are abolished by denaturation. Such optically active azochromophores provide excellent probes of local conformation and reveal its relevance to function (Fairclough and Vallee, 1970, 1971; Vallee et al., 1971).
CARBOXYPEPTIDASE
Azocarboxypeptidase. We have found diazotized arsanilic acid particularly useful in the study of structure-function relationships of carboxypeptidase A (Kagan and Vallee, 1969; Fairclough and Vallee, 1970). Modification with diazotized arsanilic acid can be followed through measurement of the number and nature of the residues by both amino acid and spectral analyses and by quantitative determination of the arsenic introduced into the protein. Under suitable conditions, treatment of carboxypeptidase A crystals with this reagent exclusively yields monoarsanilazotyrosine-248 carboxypeptidase (Johansen and Vallee, 197 1 , 1973; Johansen et al., 1972). This is of particular significance since X-ray crystallographic analysis has assigned a critical, catalytic role to Tyr-248 (Lipscomb et al., 1968). All of the arsanilazoproteins which we have studied exhibit either two or three dichroic bands with extrema between 320 and 485 nm (Fairclough and Vallee, 1970). Thus far, however, only the arsanilazocarboxypeptidase family of proteins, Le., carboxypeptidase A (Kagan and Vallee, 1969; Fairclough and Vallee, 1970; Johansen and Vallee, 197 l ) , its zymogen (Behnke and Vallee, 1971), and biologically related molecules such as carboxypeptidase S (Riordan and Livingston, 197 1) or carboxypeptidase B (Sokolovsky and Eisenbach, 1972), exhibit a distinctive absorption and circular dichroic band, in general above 500 nm and a t 5 10 nm for azocarboxypeptidase A in particular (Kagan and Vallee, 1969; Fairclough and Vallee, 1970). Therefore, the physical origin of this band was thought to differ from that of all other circular dichroic bands in this and other azoproteins (Fairclough and Vallee, 1970; Johansen and Vallee, 197 1). Our investigations have established that the azophenol group gives rise to the circular dichroic bands at 320-340 and 420-450, while that at 485 nm is due to the azophenolate ion. The unique band at 510 nm reflects the formation of an intramolecular coordination complex between the azophenol of azoTyr-248 and the active site zinc atom (Johansen and Vallee, 1971, 1973). Thus, in carboxypeptidase A a diazotized arsanilic acid reactive tyrosyl residue and the active site zinc atom are suitably juxtaposed so that an intramolecular azophenolate-metal complex can form. The Molecular Basis of Spectra of Azophenol- Metal Complexes. A number of observations and considerations demonstrate that an azoTyr-248 Zn complex indeed is the molecular basis of the absorption and circular dichroic bands at 5 10 nm. It is known that heterocyclic and aromatic azophenols form stable complexes with Zn2+ and other metals. The specific wavelength and extinction of the intense absorption maxima, generally also above 500 nm, are a function both of the metal and of the complexing agent (Anderson and Nickless, 1967; Vallee et al., 1971). In particular, arsanilazo-N-acetyltyrosineamide and tetrazolylazo-N-carbobenzoxytyrosine and their ionized species (Figures 3 and 4) can be taken as excellent models, representative of the spectral characteristics of azocarboxypeptidase A (Figure 1A,B). The formation of metal coordination complexes and their attendant spectra are characteristically p H dependent. In this regard, the consequences of absorption-pH-titrations of the T A T Zn complex and of azoTyr-248 carboxypeptidase are remarkably similar. Tridentate azophenol-metal complexes such as T A T Zn are water soluble and, hence, much more suitable for titrations over a broad range of p H than are the corresponding bidentate azo compounds. In the present study, titrationswere carried out from p H 3 to 11 to detail the physical-chemical basis of the spectra of the
-
BIOCHEMISTRY. VOL.
14,
NO.
4,
1975
655
JOHANSEN AND VAL1~t.E
T A T . metal complexes and to define the range of effectivcness of their probe properties. These data, in turn, pertain to the intramolecular arsanilazoTyr-248 Zn complex of zinc azocarboxypeptidase. Hydrogen ion titrations of T A T complexes with Zn2+, Cd2+, Hg2+, and Mn2+ result in spectral maxima and values of pK,,, for their formation and dissociation characteristic of each metal (Figure 6 and Table I). In each case, there is a bell-shaped titration curve with one pK,,, below 8.1 and another above 9.4. Analogous data have been obtained by substituting Cd2+, Hg2+, or Ni2+ for Zn2+ in azocarboxypeptidase (Johansen et al., 1973; Legg et al., to be published). Both absorption and circular dichroism-pH titrations of the azoenzyme demonstrate two pK,,, values, 7.7 and 9.5, characterizing the formation and dissociation of an arsanilazotyrosine-248 Zn complex (Figure IA). In the absence of zinc, p H
